我国登革2型病毒prM基因与甲病毒载体重组RNA的构建

Construction of the recombinant RNA of Chinese Dengue 2 virus prM gene alphavirus vector

目的 将我国登革 2型病毒的prM(D2 prM)基因导入甲病毒载体 (pSfV) ,研究该基因及其编码蛋白介导的抗病毒作用。方法 首先将扩增的病毒prM基因导入pSfV的Sp6启动子下游并进行核苷酸序列测定。用SpeⅠ酶将pSfⅤ prM重组DNA线形化 ,再将其体外转录成 5′末端含帽子结构的重组RNA。然后通过电穿孔法将此重组RNA转染BHK 2 1/ 13细胞。采用X Gal原位染色和免疫荧光法对转染细胞的表达产物进行分析。结果 通过碱基序列测定证明病毒的prM基因已插入到甲病毒载体中 ,而且其重组DNA的转录物经RNaseA消化证实为RNA。重组RNA的细胞转染率达 90 %以上。prM蛋白表达细胞约占 80 %。结论 由pSfV prMRNA转染哺乳动物细胞所产生的蛋白可与登革 2型病毒多克隆抗体起特异反应 ,证明体外转录的重组RNA含有登革 2型病毒特异序列。

Objective The aim of this study is to insert the premembrane (prM) gene of the Chinese Dengue 2 virus into the alphavirus vector (pSfV) and to investigate the prM gene and its encoding protein mediated resistance to Dengue virus. Methods The amplified prM gene was cloned into the Sp6 promoter downstream of pSfV vector and its nucleotide sequence was determined. The recombinant plasmid DNA was linearized by the enzyme Spe I digestion and transcribed in vitro into recombinant RNA which contained the capping analog on its 5′ end. Then the BHK 21/13 cells in vitro was transfected with synthesized RNA by electroporation. Analysis of the expressed products in transfected cells was performed with in situ X Gal staining and immunofluorescence technique. Results The nucleotide sequence assay of the fragment inserted in the recombinant plasmid DNA indicated that the viral prM gene had been cloned into the alphavirus vector. Assay with RNase A digestion showed that in vitro the synthesized transcripts from the recombinant pSfV prM DNA was RNA. Transfection efficiency of the pSfV prM RNA was greater than 90% and the number of positive cells expressing prM protein was 80%. Conclusion The expressed protein in mammalian cells transfected with the recombinant pSfV prM RNA can react with polyclonal antibody against Dengue 2 virus, indicating that the in vitro transcribed recombinant RNA contains specific nucleotide sequence of Chinese Dengue 2 virus strain.